JPS6080768A - Method and reagent for measuring basic fetal protein - Google Patents

Abstract

PURPOSE:To enable measurement of basic fetal protein with good accuracy by selecting the antibasic fetal protein monoclonal antibodies reacting respectively with different antigenic determinants as two antibodies. CONSTITUTION:The monoclonal antibasic fetal protein antibodies reacting respectively with the different antigenic determinants of basic fetal protein as two antibodies are selected and the basic fetal protein is measured by a sandwich method. For example, the basic fetal protein is first immunized three times to a rabbit and the blood is drawn therefrom. The serum is separated from the same and after salting out, the serum is refined by an affinity column chromatography using the basic fetal protein to obtain the polyclonal antibasic fetal protein. The measurement using the monoclonal antibasic fetal protein antibody and the measurement using the polyclonal antibasic fetal protein antibody are accomplished with all the specimens and a BFP value is determined. The two BFP values obtd. in such a way have a good correlative characteristic and the result equal to that when a polyclonal antibody is used is obtd.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method and reagent for measuring basic fetal protein.

Basic fetal protein (Bavlc Fetoprotein)
: Abbreviated as BFP) has a molecular weight of 73,00 and was discovered by one of the inventors in human fetal serum, intestine, and brain tissue.
0, a basic protein with an isoelectric point of 9.3 that migrates to the γ-globulin region in electrophoresis. This protein is specifically referred to as basic fetal protein because it is basic, in contrast to known fetal proteins that are acidic. Furthermore, one of the inventors of the present invention established a radioimmunoassay for the protein and learned that measuring the protein in serum is useful for diagnosing cancer and determining its disease course and therapeutic effects. In particular, it was learned that the protein is not useful for diagnosing cancer in a specific organ like α-phnitoprotein, but is useful for differential diagnosis between non-cancer and cancer, that is, for diagnosing the presence or absence of cancer.

Documents (1) to (7) listed below describe the above-mentioned conventional knowledge, and their descriptions are cited as reference information for the present invention.

(1) Masaru Hoben et al.: Feto-Neoplasti
c Research on Antigen, article from the 34th Annual Meeting of the Japanese Cancer Society, p. 173 (1975).

(2) Masaru Houben: New fetal protein b present in various malignant tumors
Research on asic fetoprotein, History of 1 missing character, 100(3), 344-346(19
77).

(3) Masaru Houben: Peisic Fetoprotein (Basic Fetal Protein), History of Medicine, 106(5), 273
-281 (1978).

(4) l5hii, M: A new car
cinoembryonic protein cha
-racterized by basic prop
erty, 5candinav, J.

Immunol, 8 (Suppl, 8), 6
11-620 (197B).

(51l5hii, M.: Characteri
zation of basic fetoprote
inVol, 1. Lehmann, F.-G.
(ed.), Elisevier/North-Ho
lland Biome Cal Press, Am
sterdam, 333-340 (1979).

(6) l5hii, M,, NishN15hi,
K., Hattori, M., Kanda.

Y, and l5hihara, A, : Po5
toperative 5urveillancein
patients with stomach ca
cer and monitoring of imm
uno-and polychemotherapy
in patients with leukemia
by basic fetoprotein, Car
cino-Em-bryonic Proteins,
Chemistry, Biology, Cln1
calApplication, Vol, 2 Le
hmann, F.-G. (ed.), Eli-
sevier/North -Holland Bio
rnedical Press, Amster-da
m, 603-606 (1979).

(Power l5hii, M, : C11nical u
sefulness of basic fetopr
o-Research, Herberman, R.
B, (ed,), Vol, 1゜Elisevie
r/North-Houand Biomedical
Press, New York, 45-50 (1
979).

Now, since the amount of basic fetal protein present is minute even in the serum of cancer patients, it is necessary to establish a highly sensitive measurement method for its quantification, mainly RIA method and EI method.
Method A has been developed. For details of the RIA method, the descriptions in the above documents (3) to (5) are cited as reference information. For details of the EIA method, see the following document (8).
The descriptions in ~(9) are similarly cited as reference information.

(8) Masaru Hoben and others: Ba5ic Fetopro
tein, Modern Clinical Functional Tests - Their Practice and Interpretation 1 Japan Clinical 37: 1536-1539.
Fetoprotein), Clinical Test 24 (8):
931-936゜1980゜After that, the antibodies used by the present inventors were mass-produced.

A monoclonal anti-basic fetal protein antibody was prepared for the purpose of further increasing the accuracy of the above-mentioned RIA++- and EIA methods using the antibody.

Regarding the production and results of the monoclonal antibody, the description in the following document (10) is cited.

(1 ■ 10th Japanese Society of Clinical Immunology (Osaka) lecture abstract p.
193 (1981), Preparation of Monoclonal Antibodies against Basic Fetal Proteins The reaction specificity of the monoclonal antibodies obtained here against basic fetal proteins was investigated using the EIA method and the MO method. As a result, it is known that there are at least three different types of antigenic determinants on the basic fetal protein molecule, and that monoclonal antibodies can be classified into three types according to each antigenic determinant. reached. The three types of antigenic determinants are A, B, and C, and the monoclonal antibodies corresponding to each antigenic determinant are listed as follows.

Antigenic determinant Monoclonal antibody A 5C,4,5C5,5C6,7DI, Klf2.
5B2.5B3.502 Therefore,9 the present inventor conducted various studies on methods for accurately measuring basic fetal protein by the Sand-Deutsch method using these monoclonal antibodies.As a result,
It has been learned that a combination of monoclonal antibodies that react with different antigenic determinants should be selected as the two antibodies used in the Sand-Deutsch method. For example, if selected from among the monoclonal antibodies exemplified above, a combination of 5C4 and 5C2 or a combination of 1 and 5
Examples include a combination of B3.

The present invention was completed for the measurement of basic fetal protein based on this knowledge.

In other words,9 the purpose of the present invention is to enable accurate measurement of basic fetal protein using the Sand-Deutsch method.9
To achieve this objective, the present invention discloses a technique for selecting anti-basic fetal protein monoclonal antibodies that react with different antigenic determinants as two antibodies in the Sand-Deutsch method.

The present invention will be explained in detail below.

The basic fetal protein measured by the present invention has a molecular weight of 7.
3,000, and is a basic protein that migrates to the γ-globulin region in electrophoresis and has an isoelectric point of 9.3,
As mentioned above, it is a tumor marker with low organ specificity.

The anti-basic fetal protein monoclonal antibody according to the present invention may be produced, for example, as follows. First, antibody-producing cells were prepared from BALB/C mice sensitized to basic fetal protein, and P3-X63-Ag8 myeloma cells were prepared.
- Cells are fused with Ul by a modified method of Herzenberg et al. Next, the antibody titer from the fused cells selected in HAT medium is measured by plate binding assay using 125-labeled basic fetal protein. The fused cells exhibiting high antibody titers and good cell proliferation are cloned by limiting dilution to obtain several types of cell lines that produce high antibody titers. Finally, each cell line is transplanted into the peritoneal cavity of a mouse, the obtained ascites is salted out with ammonium sulfate, and after dialysis, the IgG components are collected by DBAE-cellulose, and several types of monoclonal antibodies according to the present invention can be obtained. can. In order to classify the obtained monoclonal antibodies according to different antigenic determinants of basic fetal protein, the following MO method and E method are used.
Just implement the IA method.

MO method Prepare a mixture of two types of monoclonal antibodies at a ratio of 1:1 for all monoclonal antibody combinations, and use the MO method to generate a sedimentation line between these mixtures and basic fetal protein. Also observe the presence or absence of fusion. If a clear sedimentation line is observed, the monoclonal antibodies involved in the combination belong to a group that reacts with different antigenic determinants; 9On the other hand, if no sedimentation line is observed, they belong to a group that reacts with the same antigenic determinant.

If the generation of the sedimentation line is unclear and the 2nd judgment is uncertain, the 9th order EIA method is used to judge.

All of the measurement reagents prepared in the same manner as in Example 1 except that an arbitrarily selected monoclonal antibody with an L-terminus was used instead of 1 and 5C2 in EIA method Example 1 were used. Prepare a combination of monoclonal antibodies. E.I.
Perform operation A and read the coloring value 0D4n and affi. If there is coloration, the combination will be affected by the combination. −
7J-+J y JM ffJ-+ m JP 2 F
#Ii! fnh11: belongs to the +r reaction group9
On the other hand, if there is no color development, they belong to a group that reacts with the same antigenic determinant.

When grouping is performed using the MO method and the EIA method, as mentioned above, basic fetal protein has three different antigenic determinants, and monoclonal antibodies are classified into three groups that react with each of them.

Next, the measuring method of the present invention will be explained.

The measurement method of the present invention is a Sand-Deutsch method using two antibodies, and utilizes either an enzyme immunoassay or a radioimmunoassay. The usual procedures in these methods are already generally known per se. Therefore, the measurement method of the present invention will be explained as follows when using enzyme immunoassay, for example.

The components of the entire measurement system are solid phase, solid phase coating antibody (first antibody), basic fetal protein (standard antigen and test serum),
These are a labeling antibody (secondary antibody), an enzyme, and a substrate. As the solid phase, wells of a microtiter plate for enzyme immunoassay may be used. Prior to measurement, one type of monoclonal antibody according to the present invention is arbitrarily selected as the antibody for solid phase coating (first antibody), and O
D...

0.1M so that it becomes 0.050! The solid phase surface is coated with the first antibody by dissolving it in J-phosphate buffered saline (pH 7.2) and placing it in a well for enzyme immunoassay made of polystyrene, for example, and leaving it at 4°G overnight. The standard antigen used was purified basic fetal protein prepared by purifying ascites obtained from a liver cancer patient, and adjusted to a predetermined concentration using a standard antigen dilution solution.

As the labeling antibody (second antibody), a monoclonal antibody according to the present invention that reacts with an antigenic determinant different from that with which the first antibody reacts is selected. An example of an enzyme is alkaline phosphatase.

Glucose oxidase, peroxidase, beta-galactosidase, etc. can be used.

Prior to measurement, the labeled antibody is bound to the enzyme using a binding agent such as geltaraldehyde. The gate can be prepared in advance as part of the reagent for carrying out the measuring method of the present invention. The substrate may be used as appropriate depending on the enzyme selected. For example, when alkaline phosphatase is selected as the enzyme, p-nitrophenyl phosphate or the like may be used.

The measurement may be carried out according to the usual procedure for enzyme immunoassay. Therefore, as shown in the Examples below, a standard antigen or test serum is added to a cup coated with the first antibody and incubated, and then an enzyme-labeled antibody 1, for example, a second antibody---an alkaline phosphatase conjugate is added and incubated. Finally, substrate 2, such as p-nitrophenyl phosphate, is added and incubated, and the amount of substrate decomposition is measured using a spectrophotometer.

The first antibody to be coated on the solid phase may be a single type of monoclonal antibody, or may be a mixture of multiple types of monoclonal antibodies.

In short, it is sufficient that the first antibody and the second antibody react with different antigenic determinants, and each antibody is composed of a single type of monoclonal antibody, or is composed of multiple types of monoclonal antibodies ( It is not a requirement that the present invention should be particularly limited as to whether or not this is accomplished.

The measurement method of the present invention gives results equivalent to those obtained when polyclonal antibodies are used in terms of color development values of standard antigens and basic fetal protein levels (hereinafter referred to as BFP values) in normal human serum, as shown in the experimental examples below. It is characterized by its ability to

Next, the measurement reagent of the present invention is a reagent that is directly used in carrying out the measurement method of the present invention, and achieves the same purpose as in the measurement method. Therefore, either an enzyme immunoassay method or a radioimmunoassay method is used based on the Sand-Deutsch method using two antibodies. Therefore, specific embodiments of the assay reagent of the present invention when using enzyme immunoassay, for example, are as follows. In other words, the two measurement reagents of the present invention are an in-phase coating antibody (first antibody) and a labeling antibody (second antibody) that react with mutually different antigenic determinants.
This is a set of essential constituents, which are used alone or in combination with any of the following optionally selected from the group consisting of a solid phase, a standard antigen, an enzyme, and a substrate. Here, if the set includes a solid phase, the solid phase is provided coated with the first antibody, or if the set includes an enzyme, the enzyme is coated with the second antibody and the conjugate. - It is free to be provided in such a state that these are also included in the aspect of the measurement reagent of the present invention.

In addition, for convenience of measurement, appropriate antigen dilution solution 9 reaction dilution solution 9 reaction stop solution, substrate solution solution method.

You are free to add buffer solutions etc. to the set.

These do not limit the invention.

The present invention will be explained in detail using the experimental examples described below.

Experimental Example 1 A solid-phase first antibody and an enzyme-labeled antibody using a sample monoclonal anti-basic fetal protein antibody and an enzyme-labeled antibody prepared in the same manner as described in Example 1 below were prepared.

In addition, a solid-phase first antibody using a polyclonal anti-basic fetal protein antibody and an enzyme-labeled antibody were prepared as follows.

That is, first, a rabbit was immunized with basic fetal protein three times, blood was collected, serum was separated, salted, and purified by affinity column chromatography using basic fetal protein to obtain polyclonal anti-basic fetal protein. Next, a part of it was added to 0.1 MPB (pH 7,2) to adjust the protein concentration to 0.
D28ofi was dissolved to a concentration of 0.05, injected into the wells of a microtiter plate for enzyme immunoassay, and after overnight release of F4, the liquid was drained, washed with deionized water, dried in a cup dryer, and solidified. This was used as the phased first antibody. In addition, other parts of the polyclonal antibasic fetal protein and alkaline phosphatase were mixed at a concentration of approximately 1π9/lrd, and 2% glitaraldehyde was added to a final concentration of 0.2%.
Add slowly so that

Dialyze against 0.05M) Lis-HCl buffer (pH 8,0) and add 100% NR8 to a final concentration of 25%.

0.05M) Lis-HCl buffer (pH 8, 0, 10% N
R8, 1mM MgCI2 (containing 0.9% NaCl) was adjusted to an optimal concentration to prepare an enzyme-labeled antibody.

In a system using a monoclonal anti-basic fetal protein antibody, a reaction dilution solution prepared in the same manner as described in Example 1 was prepared.

In addition, in a system using a polyclonal anti-basic fetal protein antibody, one prepared as follows was prepared. That is, normal rabbit serum, EDTA3Na, and sodium chloride were each added in 0.1M carbonate buffer (pH 9,0).
2% (20% when diluting standard antigen), 1
It was prepared by containing it at 0mM and 0.9%. Substrate Solution 2 The same reaction stop solution as in Example 1 was prepared. The samples were 180 normal human sera and 19 cancer patient sera for 771 samples.

Method For all samples, measurements using monoclonal anti-basic fetal protein antibodies and measurements using polyclonal anti-basic fetal protein antibodies were performed as follows.
P values (ng/mL) were calculated.

First, the sample and standard antigen of known concentration were diluted 13 times with reaction diluent, and 100μ
and incubated at 37° C. for 1 hour. Wash with purified water and then add 100% enzyme-labeled antibody at the optimal concentration.
μt was added and incubated at 37° C. for 1 hour.

Wash again with purified water, add 100 μL of substrate solution, and
The cells were incubated for 1 hour at C. lN-NaOH1
The reaction was stopped by adding 00rnl, 0D4o, fi□ absorbance values were measured, and the BFP value was calculated.

Results The results are shown in Figure 1. In Figure 1, the horizontal axis shows the BFP value obtained by measurement using a monoclonal anti-basic fetal protein antibody, and the vertical axis shows the BFP value obtained by measurement using a polyclonal anti-basic fetal protein antibody9 FIG. 1 is a graph showing the correlation between both BFP values. The equation of the regression line in the figure is y-1,016x -t-0
, 06, and the correlation coefficient is 0.981. That is, both BFP values have a good correlation, and it is therefore clear that the present invention can provide results equivalent to those obtained when polyclonal antibodies are used.

The present invention will be explained in more detail with reference to Examples described below.

Example 1 Mouse ascites containing two types of monoclonal antibodies that react with different antigenic determinants of basic fetal protein, 1 and 5C2. Saturated ammonium sulfate (4.05M) was added to each 5TLL to a final concentration of 1.8M. and stir at room temperature for 2 hours. 12. Centrifuge at OOOrpm for 20 minutes to separate the precipitate and two supernatants. The precipitate was dissolved in 0.01 M PBS (
pT48.0) in 3 ml, and dialyze the precipitate using the buffer solution in which it was dissolved. Next, Ig was applied to a DEAE-cellulose column with 0.1M PB (p+48.0).
Elute G component. The eluted IgG component was concentrated using a collodion pack (MW 75000).

Prepare K1 and 5C2. K1 at 0.1 MPH (p
H7,2) as protein concentration 0D211Q my 0.0
5, and injected into the well of a microtiter plate for enzyme immunoassay. After leaving overnight, drain, wash with deionized water, dry in a cup dryer, and solidify. Prepare the first antibody.

On the other hand, 5C2 and alkaline phosphatase (500
0 U/rnL) respectively are dialyzed against 0.075 M PBS.

IgG component and alkaline phosphatase are about 1 y? 9
Mix to a volume of 7 ml, and then slowly add 2% geltaraldehyde using a microsyringe to a final concentration of 0.2%. After reacting at room temperature for 30 minutes,
Dialyzed against 0.05M) Lis-HCl buffer (pH 8,0),
Add 100% NR8 to a final concentration of 2596 and lyophilize after assay to prepare an enzyme-labeled antibody.

Separately, prepare a reaction dilution solution, substrate solution 2 reaction stop solution, and standard antigen dilution solution as follows.

The reaction dilution solutions were normal rabbit serum, normal mouse serum (inactivated at 6°C for 30 minutes), EDTA3Na, thorium chloride, and sodium triazide in 0.05M Tris-HCl buffer (pH 8.0). 11.8%, 2%, 10
It is prepared by containing it at a concentration of mM, 0.15M, and 0.1%.

The substrate solution was 0.05 p-nitrophenyl sulfate.
M carbonate buffer (pI-19,4, IvigCl, 2
Prepared by containing 4 μL in mM (containing mM).

1N-NaOH is prepared as a reaction stop solution.

The standard antigen dilution solution is normal mouse serum (inactivated 568C
930 min), EDTA 3 Na, sodium chloride, sodium triazide 0.05 M) each 18.1%, 10 mM, 0.05 M) in Lis-HCl buffer (pH 8,0).
15M, 0.1';'6.

The above-mentioned in-phase first antibody, enzyme-labeled antibody 9 reaction dilution solution, substrate solution 9 reaction stop solution, and standard antigen dilution solution were added with lyophilized ascites from a liver cancer patient as a standard antigen to form a set containing two assay reagents of the present invention. do.

Example 2 Monoclonal antibody 5C2 is dissolved in pH 7,5,0,05M phosphate buffer and the protein concentration is adjusted to l'f//m,L. 12" of 1 milliKyurie for 10 μL of this solution.
Add 50μl of l-Na and add chloramine T nif.
Add 10 u, l of a solution dissolved in the above phosphate buffer at a ratio of 1.5"F/ml (7) and stir for 30 seconds. Next, add 2"jhr of sodium metabisulfite to the above phosphate buffer.
The reaction is stopped by adding 100 μt of the solution dissolved at a ratio of tl. Add potassium iodide to the phosphate buffer for 10~
100 .mu.l of lysate dissolved at a ratio of /rrtl was added to this reaction solution, and immediately the 125.mu. labeled substance and +2'I were separated by gel filtration using Sephadex G-50. The specific radioactivity of the obtained 12'■ labeled substance is approximately 5-201
tCi/μg.

This 1-cho-labeled second antibody is combined with the immobilized first antibody prepared as described in Example 1 to form a measurement reagent of the present invention using radioimmunoassay.

[Brief explanation of drawings]

FIG. 1 corresponds to FIG. 1 described in the section of Experimental Example 1 Results. BFP value obtained by measurement using monoclonal anti-basic fetal protein antibody and 13FP obtained by measurement using polyclonal anti-basic fetal protein antibody
It is a graph showing correlation between values. Patent application artificial - Zai Co., Ltd.

Claims (1)

[Scope of Claims] K) In measuring basic fetal protein by the Sand-Deutsch method using two antibodies, the two antibodies are monoclonal anti-basic fetal protein that react with different antigenic determinants of basic fetal protein, respectively. Method for measuring basic fetal protein characterized by selecting antibodies (2) In a reagent for measuring basic fetal protein by the Sand-Deutsch method using two antibodies, the two antibodies are used to determine different antigens of basic fetal protein. A reagent for measuring basic fetal protein, characterized by selecting a monoclonal anti-basic fetal protein antibody that reacts with each of the following groups: